SYBR Green Dye Research Articles

Down-regulation of IRF3 expression in Relapse-Remitting MS patients

Background: Relapsing-Remitting (RRMS) is the most common Multiple Sclerosis disease course. Interferon regulatory factor 3 (IRF3) as major regulators of immune system genes plays a critical role in the activation of type I interferons promoters, in particular IFNβ promoter. Hence we aimed to evaluate the expression rate of IRF3 in RRMS patients under different type of IFNβ treatment. Material and methods: In the present study total of 100 subjects participated. Blood samples of 25 patients with RRMS newly diagnosed who have not been treated with interferon components, 25 patients with RRMS treated with Interferon beta-1α (B1a), 25 patients with RRMS treated with Interferon beta-1β (B1b) and 25 control samples were collected. The samples were transferred at standard conditions to the Cellular and Molecular Research Center of Shahrekord University of Medical Sciences, RNA was extracted and converted to cDNA. To evaluate the expression of IRF3 the Real-Time PCR method using SYBR Green dye was done. The level of gene expression was measured by a comparative threshold cycle formula. The obtained data were analyzed using SPSS v15 software. Results: In the study we compared the IRF3 mRNA expression of all subjects in association with gender, which no significant difference was seen (P > 0.05). Also assessment of the gene mRNA level in study groups revealed that the B1b, B1a and new case group had the lowest expression respectively. Moreover, comparison of the mRNA level between new case and B1b groups showed remarkable difference (P 0.05). Conclusion: Perhaps the IFNβ recombinants decreases the IRF3 expression as a negative feedback mechanism. Overall the data reported here, supports the previous studies in important role of IRF3 in autoimmune inflammatory disease of CNS and Multiple Sclerosis.

Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay.

Loop-mediated isothermal amplification (LAMP) is one recently developed gene amplification technique that emerges as a simple and quick diagnostic tool for early detection of nucleic acid targets. The LAMP technique works on the principle of strand displacement activity of Bst polymerase. It contains a set of four specially designed primers, which recognizes six different regions on the target nucleotide sequence. In the LAMP reaction, amplification is carried out in an isothermal conditions (60-65°C) using simple and inexpensive device like water bath or dry bath. Additional benefits of LAMP technique are that final results can be seen directly with naked eyes by adding intercalating dye SYBR Green I in the reaction tube. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is one of the novel techniques used for detection of RNA targets. The technology has been successfully applied for rapid and sensitive detection of Citrus tristeza virus (CTV) by using four oligo-primers, targeting a conserved coat protein gene (CPG) of an Indian CTV isolate. The result of assay is visible in naked eyes easily in the presence of SYBR Green I (100×) or on 1.5% agarose gel electrophoresis. CTV-RT-LAMP could be used away from plant pathology laboratories even in remote location.

Detection of Mannheimia Haemolytica in culture and lung tissue of lambs by real-time polymerase chain reaction assay

Mannheimia haemolytica is one of the bacterial species involved in cases of ovine respiratory complex, causing significant economic loss to sheep production worldwide. In the year 2016–17, a study was undertaken with the aim to evaluate a SYBR Green dye based real-time polymerase chain reaction (PCR) assay targeting O-sialo glycoprotein endopeptidase (gcp) gene for the detection of M. haemolytica in the DNA isolated from culture and pneumonic lung tissue of neonatal lambs. The test showed thousand times more sensitivity than conventional PCR and detected down to 100 fg of genomic DNA of pure M. haemolytica. The real-time PCR was found specific for gcp gene of M. haemolytica, as no cross reactivity was detected with a variety of known bacterial isolates characterized previously by species specific or 16S rRNA PCR. The real-time PCR was employed to screen M. haemolytica in 41 ovine lung tissues collected from neonatal lambs, which showed increased level of detection as compared to the conventional PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products of 227 bp size that showed 99–100% homology with the published sequences available in the NCBI database. It was concluded that the assay may be used as a valuable diagnostic tool for the rapid and specific detection of M. haemolytica in clinical samples.

GENE POLYMORPHISMS OF ENDOTHELIAL DYSFUNCTION, COACTIVATORS OF MITOCHONDRIAL BIOGENESIS AND PLASMINOGEN/PLASMIN SYSTEM IN THE DEVELOPMENT OF CARDIOVASCULAR EVENTS IN RHEUMATOID ARTHRITIS

Cardiovascular catastrophes are the most common cause of death in patients with rheumatoid arthritis (RA). At the same time, the mechanism of progression of atherosclerotic lesions in the vascular bed in RA, including genetic factors, remains a matter of debate. Objective : to analyze the associativity of polymorphism of the NOS3, PPARG γ , PPARGC1А, PPARGC1B and PAI-1 gene with a high/low cardiovascular risk in patients with RA. Subjects and methods . The investigation enrolled 73 patients with RA. Of them, 67.1% were at high cardiovascular risk. Ultrasonography of the brachiocephalic arteries with determination of intima-media thickness (IMT) was used as a surrogate marker for cardiovascular risk. A comparison was carried out taking into account the relevant standards. The IMT value exceeding the above parameters was regarded as the sign of a high cardiovascular risk. Single nucleotide polymorphisms in the NOS3 (rs2070744), PPARG2 (rs1801282), and PPARGC1A (rs8192678), PPARGC1B (rs7732671), and PAI1 (rs1799889) genes were investigated by a real-time polymerase chain reaction assay using the intercalating dye SYBR Green I according to the instructions of the manufacturer («Litech», Russia). Results and discussion. The frequencies of the NOS3 -786ТТ genotypes significantly differed in patients at different cardiovascular risks. Complex genotypes, the frequency of which was prevalent in the low cardiovascular risk group, were identified. At the same time, all genotypes contained the homozygous NOS3 -786ТТ genotype and the genes that were able to regulate its expression. The greatest differences between the analyzed groups were present in the complexes of NOS3-786TT:PPARGC1B 203AlaAla and NOS3 -786TT:PPARGC1B 203AlaAla:12 PPARG ProPro. Conclusion. Our analyzed gene polymorphic positions can concurrently affect cardiovascular risk in patients with RA.

GFAP expression is influenced by astrocytoma grade and rs2070935 polymorphism.

Glial fibrillary acidic protein (GFAP) is an intermediate filament that provides mechanical support to astrocytes. Rs2070935 is a single nucleotide polymorphism (SNP) located in the promoter region of the GFAP gene. The aim of this pilot study is to investigate GFAP expression at mRNA, protein levels and rs2070935 polymorphism in 50 different grade human astrocytoma samples. GFAP expression at mRNA level was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) with SYBR Green dye, whereas the translational activity of the following gene was detected using western blot assay. Furthermore, genotypes of rs2070935 were identified using qPCR with TaqMan probes. As a result, GFAP mRNA and protein expression was found to be declining with increasing astrocytoma grade (p < 0.05). A tendency was observed between increased GFAP mRNA expression and shorter grade IV astrocytoma patient survival (p = 0.2117). The rs2070935 CC genotype was found to be associated with increased GFAP translational activity in grade II astrocytoma (p = 0.0238). Possible links between rs2070935 genotypes and alternative splicing of GFAP were also observed. The rs2070935 AA genotype was found to be associated with poor clinical outcome for grade IV astrocytoma patients (p = 0.0007), although the following data should be checked in a larger sample size of astrocytoma patients.

Alu and Poet Ankyrine Detection and Quantization in Cell Free Dna of Cancer Patients

In this study, specimens of plasma were collected from 96 from patient diagnosed with cancer from Al Karama teaching hospital. Also plasma collected from 25 healthy individual as control. Cell-free DNA (cfDNA) was extracted from 1ml plasma using high pure viral Nucleic Acid Kit as an alternative to high expensive cfDNA kit. cfDNA extracted according to the manufacturer’s instructions, but without the use of carrier RNA. This represent the first recorded method of using viral nucleic acid extraction kit for collection of cell free DNA. Results indicate that all cancer samples show significant increase of cfDNA in compare with control (p 0.01). and this dramatic increase in DNA concentration provide good indicator about health condition. DNA integrity have been checked depending on amplification of Arthrobacter luteus (Alu sequence). Both small (115) and large (247) large Alu stretches have amplified using RT PCR using SYBR green dye. Results shows that integrity of DNA extracted from patients and control are suitable for any further molecular investigation, and short Alu repeats are much less abundant in health individual in compare with cancer patient of all cancer types under study. Depending on these results one can conclude that almost all DNA obtained from normal patients is finely fragmented due to apoptosis action and that’s why give very little positive. results in compare with corresponding cancer patient which release large DNA fragments resulted from necrosis and NK/Tc cells activity, which is amplifiable more efficiently. In this work another molecular study conducted to investigate the cancer specific sequences. Since we deals with different cancers type in this study, two types of prostate, ovary, testis expressed protein (POTE Ankyrine) were tested 2α and 2β. Results shows that almost all healthy group were negative for specific POTE test while cancer patient samples are positive for POTE specific amplification. This results are promising since it could be developed to be a building block of cfDNA cancer tests.

Effects of oregano essential oil on brain TLR4 and TLR2 gene expression and depressive-like behavior in a rat model.

The aim of the present study was to evaluate the effects of oregano essential oil (OEO) on the hippocampus and prefrontal cortex TLR 2/4 gene expression and depressive like behavior induced by chronic unpredictable stress (CUS). Sucrose preference and forced swim tests were adopted to examine the antidepressant effect. Control (CON), OEO, CUS, and CUS + OEO groups were used. The OEO and CUS + OEO groups received OEO (0.2 mL/kg, i.p.), CON and CUS received saline (0.2 mL/kg, i.p.), and the positive drug groups of CUS rats received fluoxetine (10 mg/kg) and diazepam (3 mg/kg) once daily for 14 days. The expression of TLR 2/4 was determined using real time quantitative polymerase chain reaction with the SYBR green reporter dye. The compositions of the OEO were determined by gas chromatography-mass spectroscopy. The main constituents were thymol (20.72%), gamma-terpinene (8.83%), borneol (8.72%), cymene (6.83%), carvacrol (6.274%), alfa-terpinene (5.26%), and sabinene (4.92%). Administration of OEO significantly alleviated the depressive symptoms of CUS. A higher level of TLR2/4 mRNA was seen in the brain of CUS group (P < 0.05). The CUS-induced increases in the TLR2/4 levels were not reversed by OEO. According to the present study OEO may have the antidepressant-like activity but have no effect on the stress-induced TLR-2/4 upregulation.

Molecular Characterization of Mycoplasma Synoviae Isolated From the Respiratory System and Joints of Chickens with Special Reference of vlhA Gene

The present work was designed to make sequencing of M. synoviae isolates for identification of nucleotide differences in the M. synoviae vlhA gene which isolated from the respiratory system and Joint from chickens. A total of 224 chicken samples (109 respiratory samples, 115 joint samples) collected from birds from different flocks showing respiratory manifestation and arthritis were examined. Identification of M. synoviae by PCR Using primers corresponding to the single copy preserved 5´ end for vlhA genetic factor, amplicons for 350~400 bp then, generated which 6 and 26 out of 224 samples were positive samples for M. synoviae from culture and tissue by PCR respectively. High-resolution melting curve analysis (HRM) for amplicons using SYBR green fluorescent dye of 9 selected isolates (5 respiratory isolates and 4 arthirtic isolates) showed that different Melting temperature curves were ranged between 84.6-85.1°C for isolates MS from the respiratory samples and other melting temperature curves ranged between 73.6-74.6°Cfor isolates Ms from the arthirtic samples. The results proved that, there was a broad concordance among nucleotide sequence of all isolates which isolated from the respiratory system except in one sample which observed the point mutations and the frame-shift mutation in some nucleotide differ than other respiratory isolates in addition there is complete concordance between nucleotide sequence of all isolates which isolated from joint. However, in comparison the Respiratory M. synoviae isolates with M. synoviae joint isolates, there was clear variation in nucleotide sequence which was confirm the result of HRM. Compared to vaccine MS-H strain arrangement, all isolates show clear variation from (MsH) vaccine strain. This study proved a difference between M. synoviae isolated from the respiratory system and Joint and live commercial vaccine (MsH) strain. Also, these informations showed that variations in the vlhA gene sequence could be introduced into the expressed vlhA gene amino acid codons and effective in pathogenesis rate in herds.

Development of a SYBR Green II Real-Time Polymerase Chain Reaction for the Clinical Detection of the Duck-Origin Goose Parvovirus in China

Objective: To establish an efficient, convenient and quantitative method for the clinical detection of the duck-origin goose parvovirus. Method: In the present study, a real-time polymerase chain reaction (PCR) method was established for detecting the duck-origin goose parvovirus using the fluorescent chimeric dye SYBR Green II. Specific primers were designed to target a highly conserved region of the VP3 gene of the duck-origin goose parvovirus. Results: This method was able to detect a minimum of 19.6 copies/μL of viral genomic DNA. Results showed that this method was faster and had a higher sensitivity than the traditional PCR in the clinical specimen test. In this paper, we developed a rapid, sensitive detection and quantitative analysis technology for the duck-origin goose parvovirus by real-time PCR assay. Conclusion: This test provides improved technical support for studies regarding the clinical diagnosis and epidemiological investigations of the duck-origin goose parvovirus.

Ultrasensitive, label-free detection of T4 ligase and T4 polynucleotide kinase based on target-triggered hyper-branched rolling circle amplification

Ligase and polynucleotide kinase (PNK) play important roles in DNA repair process. In this work, a simple, convenient and ultrasensitive fluorescence biosensor for label-free detection of ligase and PNK was proposed on the basis of target-triggered hyper-branched rolling circle amplification (HRCA). In the presence of PNK and ligase, the 5′-OH end of a linear Padlock DNA strand could be phosphorylated by PNK and then be converted by ligase into a circular template to trigger subsequent exponential HRCA reaction, producing amounts of double-stranded DNA (dsDNA) fragments, accompanied by the significant fluorescence increase of dsDNA-intercalating dye–SYBR Green I (SG I). In the absence of ligase or PNK, however, HRCA primer and uncyclized Padlock strands were completely digested by exonucleases, thus providing a low background for the sensing system. Therefore, by recording the fluorescence change of SG I, ligase and PNK activity could be facilely determined. The proposed biosensor exhibited excellent detection sensitivity for ligase and PNK activity with the detection limits of 3.4 × 10−4 U/mL and 3.8 × 10−4 U/mL, respectively. Such a sensing strategy could be easily extended to studies on inhibitors of these two enzymes, thus might offer a promising tool in clinical molecular diagnostics and cancer therapy.

Validating the Efficiency of a Simplex PCR and Quantitative SYBR Green qPCR for the Identification of Salmonella spp. DNA

This study aimed to perform a comparative validation of the efficiency of quantitative SYBR Green qPCR and simplex PCR identification of Salmonella spp. DNA. For this, the samples of DNA from the Salmonella typhimurium were diluted up to 10-5 in duplicate. The results showed that the same primers were effective for both simplex PCR and qPCR. It was possible to detect the bovine species up to a dilution of 10-1 using simplex PCR. For all dilutions, it was possible to obtain qPCR amplification with a minimum Ct value of 15.13 for the 10-1 dilution for Salmonella spp. Next, the SYBR Green qPCR amplicons were separated using agarose gel electrophoresis for confirmation of the amplified fragment size. The superiority of qPCR over multiplex PCR was validated in terms of sensitivity, even with the use of SYBR Green dye, suggesting the possible use for quality control in foodstuffs.

Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis

Objective: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. Materials and Methods: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. Results: Melting temperatures (T_m) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, T_m values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. Conclusions: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening.

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.

Performance Evaluation of a Novel Sample In-Answer Out (SIAO) System Based on Magnetic Nanoparticles.

In recent years, the prevention, control, diagnosis and treatment of infectious diseases has become a global focus for public health. Traditional methods for pathogen testing have some major disadvantages including the need for highly skilled staff and expensive instrumentation, while procedural aspects are complex and sensitive to the environment. These shortcomings have greatly limited the application of traditional testing in on site pathogen detection. In this paper, we present a new point-of-care-testing (POCT) system based on magnetic nanoparticles that enable sample in-answer out (SIAO) automated real-time testing for pathogens. Various performance tests were conducted on the instrument. Nucleic acid extraction efficiencies of SIAO versus manual systems were 95.49% and 84.33%, respectively. Real-time PCR by two methods (TaqMan-based probe and SYBR green dye) in the SIAO system was achievable, with comparable results to the manual method. Nucleic acid testing with the SIAO system was repeatable and better than with manual testing. The SIAO system had good anti-pollution performance with easy avoidance of inter-assay cross contamination. Finally, use of the SIAO system for adenovirus detection produced similar results to LightCycler2.0 system assay findings. The amplification plots and Ct values suggested similar amplification plots shapes for adenovirus testing with the SIAO system and with real-time fluorescence PCR testing and commercial instrument post-manual nucleic acid extraction. Collectively, these findings indicate that testing with the SIAO system is virtually equivalent to that of manual extraction with commercial system testing.

Real-time PCR assay for rapid, efficient and accurate detection of Paramyrothecium roridum a leaf spot pathogen of Gossypium species

Here we report a highly sensitive real-time PCR (qPCR) assay to detect Paramyrothecium roridum from pure culture and infected samples of cotton plants. A specific set of primer pair pMyro F/R is designed to target the 185 bp ITS region of rDNA of Paramyrothecium roridum species and validated using qPCR. The fluorescence signals were detected above the baseline threshold from samples containing Paramyrothecium roridum DNA, whereas other samples did not produce any fluorescence or produced fluorescence which did not reach detection threshold values. A single dissociation peak of increased fluorescence was obtained for the specific primers at 92.2 °C melting temperature. The limit of detection using SYBR Green dye in this assay was up to 0.1 pg per µL of DNA from pure culture of P. roridum. The assay is accurate, sensitive, less laborious and time saving for detection of P. roridum in infected tissues of cotton.

The sensitivity and efficacy method of PIK3CA exon 9 E545A as a high diagnostic accuracy in breast cancer.

The phosphatidylinositol 3-kinases (PIK3s) are lipid kinases. Mutation in the exon 9 and exon 20 determined as a predictive factor in anti-HER-2 therapy. In some countries, such as Singapore, China, and Peru, PIK3CA exon 9 E545A was reported to produce the highest rate of mutation. In this research, we developed and optimized PIK3CA exon 9 E545A detection methods with intercalating dye SYBR Green I based on the Tm Shift approach by using prepared recombinant plasmid pGEMT-easy PIK3CA exon 9 and PIK3CA exon 9 E545A. Recombinant plasmid was used due to the limited number of samples.MethodsRecombinant plasmid was prepared based on manufactured procedures, and this process was then followed by Tm prediction with Poland software, Tm Shift SYBR Green I development, and its characterization (reproducibility, repeatability, sensitivity, qPCR efficiency, and qPCR amplification), respectively.ResultA method for PIK3CA E545A detection based on TM shift SYBR Green I has been successfully developed. The melting temperature for PIK3CA exon 9 was 78.1 ± 0.1 °C, while that for PIK3CA exon E545A was 80.20 °C. The Tm of mutant was the same as that predicted using Polland Software. The reproducibility of the methods was high, with the coefficient values for inter and intra assays were below 10% with a high sensitivity at 1%, while R2 0.99 and PCR efficiency was 97.75%.ConclusionThe results presented here demonstrate that the PIK3CA exon 9 E545A detection method has a good sensitivity and efficacy assay, which proves that the method has a high diagnostic accuracy in breast cancer.

Identification and characterization of Alternaria alternata (Fr.) Keissler causing Ceratonia Blight disease of carob (Ceratonia siliqua L.) in Turkey

Carob (Ceratonia siliqua L.) leaves showing suspected symptoms of Ceratonia Blight disease were collected from both wild and domestic carob plants at various locations in the province of Antalya in Turkey. The fungus growing on culture medium started as white-grayish airy mycelium at the margin with clear light to dark green inner zonation radiating from a common center. Dark brown conidia in chains were observed ranging in sizes from 5 to 35 μm. The conidia surface was smooth to verruculose, slightly constricted with 4–6 transverse septa; the lower part of each portion had one or two longitudinal septa. The conventional PCR was conducted based on the beta tubulin gene and ITS region, which resulted in fragment sizes of 184 bp and 346 bp, respectively. The amplified PCR products were sequenced aligned and BLAST analysis showed 100% homology for the two genes with other strains from the GenBank nucleotide database. The isolates also tested positive with SYBR Green fluorescence dye using Real-Time PCR. The results of the disease incidence recorded across the fields ranged from 10 to 100% on both wild and domesticate carob plants. The putative pathogen was pathogenic on the inoculated carob plants and was consistently re-isolated from the symptomatic plant leaves and thereby satisfying the Koch’s postulates. Based on our knowledge, this is the first report implicating Alternaria alternata for causing Ceratonia Blight disease in Turkey.

Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase

Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.

Ultrasensitive microRNA-21 detection based on DNA hybridization chain reaction and SYBR Green dye

It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density. The detection method showed a wide linear region from 1 fM to 105 fM, and the limit of detection was 0.2554 fM (at S/N = 3) for microRNAs. The results showed that this method had an excellent specificity and reproducibility. Furthermore, the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method (R2 = 0.994). Overall, the proposed strategy could be satisfactory for rapid, ultrasensitive and efficient detection of microRNA-21, and held great potentials in clinic diagnosis of gastric cancer.

Expression study of CYP19A1 gene in a cohort of Iranian leiomyoma patients

BackgroundCYP19A1 gene encodes aromatase, a microsomal key enzyme that catalyzes the synthesis of estrogens from androgens. Accumulating evidence has revealed that aromatase plays an important role in the pathogenesis of leiomyoma through increasing local concentration of estrogens. In this study, we examined the levels of CYP19A1 mRNA to determine the impact of aromatase overexpression in uterine leiomyoma growth. Subjects and methodsTissues were obtained via myomectomy or hysterectomy from 30 patients. Total RNA was extracted and cDNA was synthesized from each frozen sample. Using SYBR Green dye, Real-time PCR assay was performed by sequence-specific primers. Relative mRNA expression was normalized to the mean of the Ct values determined for HPRT1. Gene expression ratio in each sample was determined relative to the mean ΔCt value of tumor-free margin samples. ResultsPCR efficiencies for amplification reactions of HPRT1, and CYP19A genes were calculated as 0.93 and 0.96, respectively. Regression coefficients (R) for standard curves were above 0.90. The obtained data revealed that the mean fold increase of CYP19A1 gene expression in leiomyoma samples relative to normal samples was 3.551 (95% CI: 0.04–6.64, S.E., 0.29–5.35). ConclusionsOur results were in accordance with previous studies and imply that up-regulation of CYP19A1 is correlated with the pathogenesis of leiomyoma tumors. We also observed that expression level of CYP19A1 was not linked to the tumor size or localization. It can be concluded that; up-regulation of aromatase is a key factor in the initiation of tumor development as well as tumor growth.